The research described in this proposal will test the hypothesis that the secretion of let-7b?a 22-nucleotide micro-RNA (miRNA)?by human bronchial epithelial cells (HBEC) in extracellular vesicles increases the antibiotic sensitivity of and reduces biofilm formation by Pseudomonas aeruginosa, and is thereby an important mechanism of host-pathogen interactions in the lungs. P. aeruginosa is an opportunistic pathogen that infects the lungs of individuals with chronic obstructive pulmonary disease (COPD), cystic fibrosis (CF), and pneumonia. P. aeruginosa contributes to 5?10% of the acute-exacerbation events that can occur with COPD, which afflicts 24 million Americans and is the 3rd-leading cause of death in the U.S. Let-7b, like all miRNAs, suppresses gene expression, is highly conserved across species, and regulates the innate host immune responses to pathogens. In preliminary studies, this research team made the unique observations that: (1) extracellular vesicles secreted by primary HBEC deliver let-7b into P. aeruginosa; and (2) that let-7b increases the ability of front-line antibiotics to kill P. aeruginosa and reduce biofilm formation by targeting genes coding for an RND efflux pump, genes essential for biofilm formation, and genes coding for ?-lactamases. The team also made the novel observation that let-7b is dramatically reduced in extracellular vesicles isolated from the bronchoalveolar fluid (BALF) of CF patients, compared to BALF from healthy subjects, leading them to conclude that let-7b secretion in extracellular vesicles is defective in CF. Aside from the clinical implications, these preliminary data are exciting because they are the first direct demonstration that a eukaryotic miRNA can regulate prokaryotic function. Building on these findings, the team propose three specific aims: (1) Test the hypothesis that let-7b increases P. aeruginosa sensitivity to fluoroquinolone antibiotics by reducing the RND efflux pump, MexGHI-OpmD; (2) Test the hypothesis that let-7b inhibits the formation of P. aeruginosa biofilms by targeting genes essential for biofilm formation; and (3) Test the hypothesis that let-7b increases P. aeruginosa sensitivity to ?-lactam antibiotics by targeting ?-lactamases. Work in this project will utilize: (a) RNA-seq and mass spectrometry to identify genes and proteins downregulated by let-7b in P. aeruginosa; (b) GRIL-seq to directly identify let-7b mRNA targets in P. aeruginosa; and (c) a co-culture model developed by the team to study P. aeruginosa biofilms growing on HBEC. Combined, this work will elucidate the mechanisms whereby let-7b, which is anti-inflammatory, enhances the ability of antibiotics to kill P. aeruginosa and reduce biofilm formation. Since several members of the let-7 family are currently in clinical trials, and because chronic lung infections of P. aeruginosa in COPD and CF are associated with a hyper-inflammatory state, the long-term goal of this work is to develop an approach utilizing let-7b in combination with antibiotics to reduce P. aeruginosa lung infections?and the accompanying hyper-inflammatory state?in patients with CF and COPD.
Our research, aimed at understanding how let-7b inhibits bacterial growth and biofilm formation, may lead to the identification of new therapeutic targets that can be manipulated to eliminate chronic P. aeruginosa lung infections in people with cystic fibrosis, chronic obstructive pulmonary disease, and pneumonia.
